Liposomal-lupane system as alternative chemotherapy against cutaneous leishmaniasis: macrophage as target cell.

Leishmania amazonensis causes human diseases that range from self-healing to diffusion cutaneous lesions. The chemotherapy of leishmaniasis requires long-term treatment and has been based on the use of pentavalent antimonials. Liposomes have been used as antileishmanial drug carries and have adjuvant activity in vaccines against several microorganisms, representing an important option to the development of new therapeutics for the disease. In this study, we developed a liposomal formulation containing lupane [3ß,6ß,16ß-trihydroxylup-20(29)-ene], isolated from fruits of Combretum leprosum with pharmacological properties as antinociceptive, anti-inflammatory, antiulcerogenic and antileishmanial activities. The aim of the present study was to evaluate the efficacy of liposomal-lupane in L. amazonensis-infection model. Liposomes were prepared by the extrusion method with DPPC, DPPS and cholesterol at 5:1:4 weight ratio. The lupane (2 mg/mL) was added to the lipid mixture, solubilized in chloroform and dried under nitrogen flow. The activity of liposomal-lupane was conducted in vitro with mouse peritoneal infected macrophages. Furthermore, mice were infected in the right hind footpad with 10(5) stationary growth phase of L. amazonensis promastigotes. After 6 weeks, animals were treated with liposomal-lupane for 15 days by intraperitoneal injection. The evolution of disease was monitored weekly by measuring footpad thickness with a caliper. Three days after the treatment, peritoneal macrophages were collected, plated and production of the cytokines IL-10 and IL-12 was evaluated in supernatants of the cultures after 24 h. The results indicate that the liposomal system containing lupane achieved here is a promising tool to confer antileishmanial activity to infected macrophages.

Using multidimensional projection techniques for reaching a high distinguishing ability in biosensing.

Recent advances in the control of molecular engineering architectures have allowed unprecedented ability of molecular recognition in biosensing, with a promising impact for clinical diagnosis and environment control. The availability of large amounts of data from electrical, optical, or electrochemical measurements requires, however, sophisticated data treatment in order to optimize sensing performance. In this study, we show how an information visualization system based on projections, referred to as Projection Explorer (PEx), can be used to achieve high performance for biosensors made with nanostructured films containing immobilized antigens. As a proof of concept, various visualizations were obtained with impedance spectroscopy data from an array of sensors whose electrical response could be specific toward a given antibody (analyte) owing to molecular recognition processes. In addition to discussing the distinct methods for projection and normalization of the data, we demonstrate that an excellent distinction can be made between real samples tested positive for Chagas disease and Leishmaniasis, which could not be achieved with conventional statistical methods. Such high performance probably arose from the possibility of treating the data in the whole frequency range. Through a systematic analysis, it was inferred that Sammon's mapping with standardization to normalize the data gives the best results, where distinction could be made of blood serum samples containing 10(-7) mg/mL of the antibody. The method inherent in PEx and the procedures for analyzing the impedance data are entirely generic and can be extended to optimize any type of sensor or biosensor.

Biosensors for efficient diagnosis of leishmaniasis: innovations in bioanalytics for a neglected disease.

The need for reliable, fast diagnostics is closely linked to the need for safe, effective treatment of the so-called "neglected" diseases. The list of diseases with no field-adapted diagnostic tools includes leishmaniasis, shigella, typhoid, and bacterial meningitis. Leishmaniasis, in particular, is a parasitic disease caused by Leishmania spp. transmitted by infected phlebotomine sandfly, which remains a public health concern in developing countries with ca. 12 million people infected and 350 million at risk of infection. Despite several attempts, methods for diagnosis are still noneffective, especially with regard to specificity due to false positives with Chagas' disease caused by Trypanosoma cruzi . Accepted golden standards for detecting leishmaniasis involve isolation of parasites either microscopically, or by culture, and in both methods specimens are obtained by invasive means. Here, we show that efficient distinction between cutaneous leishmaniasis and Chagas' disease can be obtained with a low-cost biosensor system made with nanostructured films containing specific Leishmania amazonensis and T. cruzi antigens and employing impedance spectroscopy as the detection method. This unprecedented selectivity was afforded by antigen-antibody molecular recognition processes inherent in the detection with the immobilized antigens, and by statistically correlating the electrical impedance data, which allowed distinction between real samples that tested positive for Chagas' disease and leishmaniasis. Distinction could be made of blood serum samples containing 10(-5) mg/mL of the antibody solution in a few minutes. The methods used here are generic and can be extended to any type of biosensor, which is important for an effective diagnosis of many other diseases.

Use of proteoliposome as a vaccine against Trypanosoma cruzi in mice.

We have generated proteoliposomes carrying proteins of Trypanosoma cruzi for use as immunogens in BALB/c mice. T. cruzi trypomastigote and amastigote forms were sonicated and mixed with SDS, with 94% recovery of soluble proteins. To prepare proteoliposomes, we have used a protocol in which dipalmitoylphosphatidylcholine, dipalmitoyl-phosphatidylserine and cholesterol were incubated with the parasite proteins. BALB/c mice immunized with 20microg were able to generate antibodies which, in Western blotting, reacted with the proteins of T. cruzi. We further investigated the ability of peritoneal cells from immunized mice to arrest the intracellular replication of trypomastigotes, in vitro. After 72h of culture, the number of intracellular parasites in immunized macrophages decreased significantly, as compared to controls. Despite the fact that exposure of mice to T. cruzi proteins incorporated into proteoliposomes generate antibodies and activate macrophages, the immunized mice were not protected against T. cruzi intraperitoneal challenge.

Diabete como modelo de neuropatia autonômica / Diabetes as an autonomic neuropathy model

RESUMO: Modelo do Estudo: O presente estudo é uma revisão de literatura sobre a neuropatia diabética, especialmente a neuropatia autonômica diabética, enfatizando as recentes descobertas sobre o assunto, com modelos experimentais da doença.Importância do problema: Comprometimento do sistema nervoso autônomo ocorre em uma variedade de doenças sistêmicas, das quais o diabetes mellitus é a mais comum. Alterações no sistema nervoso periférico em animais geneticamente diabéticos ou com diabete induzi da experimentalmente têm sido amplamente investigadas, na esperança de que as alterações encontradas em modelos animais possam reproduzir algumas das alterações da neuropatia diabética humana precoce, levando ao melhor entendimento sobre o mecanismo da doença. Vários agentes químicos são citotóxicos para as células beta do pâncreas porém, apenas o aloxane e a estreptozotocina (STZ) tem sido sistematicamente investigados e são amplamente empregados para induzir diabete nos animais. Em geral, STZ é mais eficaz e mais específica para as células beta do pâncreas que o aloxane. Embora estudos morfológicos tenham sido realizados em vários nervos periféricos de ratos diabéticos induzidos com STZ, tais como sural, fibular e tibial, informações sobre alterações patológicas em nervos autonômico são limitadas.Comentários: Recentemente, estudamos as alterações do nervo depressor aórtico (NDA) em ratos diabéticos agudos (15 dias após injeção de STZ). Nossos resultados mostraram características de atrofia axonal em 5 dos 10 nervos estudados. Um estudo de microscopia de luz, conduzido em nosso laboratório, o nervo vago cervical de ratos diabéticos crônicos (12 semanas após injeção de STZ), mostrou resultados discretos, a favor da perda de fibras mielínicas finas. Outro estudo recente do nosso laboratório mostrou uma redução do número de fibras mielínicas pequenas dos nervos renais, nos ratos diabéticos crônicos, resultado mais evidente que o observado no nervo vago. Por outro lado...